JAC Advance Access originally published online on May 31, 2007
Journal of Antimicrobial Chemotherapy 2007 60(2):258-262; doi:10.1093/jac/dkm171
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Characterization of the IncA/C plasmid pCC416 encoding VIM-4 and CMY-4 ß-lactamases
1 Dipartimento di Biologia Molecolare, Laboratorio di Fisiologia e Biotecnologia dei Microorganismi, Università di Siena, Siena, Italy 2 Laboratory of Bacteriology, Hellenic Pasteur Institute, Athens, Greece 3 Dipartimento di Malattie Infettive, Istituto Superiore di Sanita, Rome, Italy 4 Laboratorio di Microbiologia, Ospedale di Circolo, Varese, Italy
* Corresponding author. Tel: +30-210-6478810; Fax: +30-210-6423498; E-mail: miriagou{at}mail.pasteur.gr
Received 18 December 2006; returned 5 February 2007; revised 10 March 2007; accepted 24 April 2007
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Abstract |
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Objectives: To characterize the antibiotic resistance regions of pCC416, a VIM-4- and CMY-4-encoding plasmid from clinical enterobacteria, and to elucidate its relation with the CMY-encoding plasmids widely diffused in Salmonella.
Methods: The enterobacterial multiresistant plasmid pCC416 was derived from an Escherichia coli transconjugant and characterized. Conventional and long-range PCR assays were performed using primers specific for VIM-4- and CMY-4-encoding segments of pCC416. Amplicons were characterized by sequencing. blaVIM-4, blaCMY-4 and IntI1-specific probes were prepared from PCR products and used for the identification of various pCC416 clones. VIM- and CMY-positive BamHI and Sau3AI fragments of pCC416 were cloned into pACYC184 and their sequences were determined by gene walking.
Results: The pCC416 plasmid contained two distinct resistant loci carrying ß-lactamase genes. The blaVIM-4 gene was part of an integron located in a complex, multidrug-resistant region of novel structure, interspersed with mobile elements or remnants thereof and being similar to various regions of other resistance plasmids. Nevertheless, a region in the 3' end of this structure resembled the respective region found in a CMY-2-encoding plasmid from Salmonella. The blaCMY-4 gene was identified within an 11.3 kb region also related to the CMY-2-encoding plasmids.
Conclusions: pCC416 probably evolved from an IncA/C2, CMY-encoding plasmid through acquisition of a VIM-encoding In4-type integron providing an example of accretion of resistance determinants in a single replicon.
Keywords: metallo-ß-lactamases , cephalosporinases , multiresistance
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Introduction |
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Plasmids are major vehicles for horizontal spreading of antibiotic resistance genes among bacterial pathogens. Accumulation of various resistance genes on a replicon confers an evolutionary advantage because a single transfer event can result in resistance to multiple antibiotics in the respective host.1 The spread of pCC416, a VIM-4 metallo-ß-lactamase (MBL)- and CMY-4 cephalosporinase-encoding, multiresistant plasmid among Klebsiella pneumoniae and Enterobacter cloacae clinical strains in Italy has been described recently.2 The resistance spectrum of pCC416 included virtually all ß-lactam antibiotics as well as aminoglycosides.2 In a subsequent comparison of multiresistant plasmids from enterobacteria isolated in diverse geographic areas, it was noted that pCC416 belonged to the same incompatibility group (IncA/C2) and exhibited highly similar restriction fragment length polymorphism (RFLP) patterns with a group of blaCMY-2-carrying plasmids that have been spread among animal isolates of Salmonella from the USA.3,4 This study provides indications that pCC416 evolved from the latter plasmid type by acquisition of a novel transposon that included a blaVIM integron.
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Bacterial strains and plasmids
The previously described Escherichia coli J53 transconjugant CON036A was used as a source of plasmid pCC416.2 E. coli XL1-Blue (Stratagene Ltd, Cambridge, UK) was used as a host for recombinant plasmids. Bacteria were routinely grown in Mueller–Hinton (MH) agar. Plasmid pACYC184 was used as a cloning vector.
MICs of various non-ß-lactam antimicrobials, including streptomycin, trimethoprim, sulfamethoxazole and chloramphenicol, were determined by an agar dilution technique.
PCR primers used in this work for detection and mapping purposes are listed in Table 1, and their locations are shown in Figures 1 and 2. Long PCR assays to amplify fragments of size > 12 kb were performed using the Expand Long Template PCR System (Roche, Manheim, Germany).
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Plasmid DNA was purified by an alkaline lysis method.5 Restriction fragments of plasmid DNA were subjected to Southern-blot hybridization directly on dried gels6 using 32P-labelled probes specific for blaVIM-4, blaCMY-4 and intI1 genes. Colony-blot hybridization for screening plasmid libraries was performed on replicate bacteria grown on nitrocellulose filters layered on MH agar.5 Probes were made of PCR products using the primers described in Table 1.
Characterization of pCC416 segments containing resistance genes was carried out by a conventional cloning and sequencing approach. Plasmid fragments cloned into pACYC184 and used for this purpose are indicated in Figures 1 and 2. Clone B1 was obtained by cloning a 16.3 kb BamHI fragment of pCC416 that hybridized with a blaVIM-4 probe. Clones TOB2R, 5G3 and C12 were selected from a library constructed by cloning an Sau3AI partial digest of pCC416, based on their hybridization to an intI1 (TOB2R and 5G3) or to a blaCMY-4 probe (C12).
Sequencing of PCR products and recombinant DNA was carried out on both DNA strands following a primer walking approach at the Macrogen Inc. sequencing service (Seoul, Korea). Similarity searches and computer analysis of the sequence data were performed using programs at the NCBI site (http://www.ncbi.nlm.nih.gov/). Insertion sequence analysis was carried out online using the IS Finder Database at http://www-is.biotoul.fr/.
Nucleotide sequence accession number
The nucleotide sequences reported here have been assigned accession numbers AJ704863 and AJ875405 in the EMBL/GenBank/DDBJ databases.
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The blaVIM-4 integron of pCC416 and its genetic environment
blaVIM-4 was found as the first gene cassette of a class 1 integron, designated In416, followed by aacA7, dfrI, aadA1 and smr gene cassettes (Figure 1b). The dfrI cassette was fused with the aadA1 gene by an 81 bp deletion, involving part of the attC recombination site of the dfrI cassette and the 5' end of aadA1. E. coli CON036A and an E. coli XL1 carrying the pACYC184-cloned fragment TOB2R (Figure 1a), while resistant to trimethoprim, were susceptible to streptomycin confirming inactivation of aadA1 (data not shown). Most of the attC site of the smr cassette was also deleted. The composition and gene cassette array of In416 resembled those of previously described VIM-1-encoding integrons such as In-e541 found in E. coli7 and K. pneumoniae8 and the integron carried by the plasmid pPBH01 from K. pneumoniae (Figure 1c).9,10 The latter integron also included a dfrI–aadA1 fusion as observed for In416.9 However, these VIM-1-encoding integrons all lacked the smr cassette found in In416 (Figure 1c). Moreover, the 5'-conserved segment (CS) of In416 contained a complete intI1 gene bounded by a 25 bp IRi, whereas in the similar VIM-1 integrons, parts of the 5'-CSs were deleted because of IS26 insertions.7,10,11 The IRi of In416 was located between the resI and resII sites of a Tn1696 module, in the same configuration previously described in In4 and In34.12,13 The IRR of Tn1696 was interrupted by an IS5075 element (Figure 1b). This sequence corresponded to the region flanking In34 where an IS4321 (93% homologous to IS5075) was inserted at the same position in Tn1696,14 suggesting separate events of acquisition of these similar ISs at a hotspot located within IRR.
In416 lacked a 3'-CS typical of sul1-associated integrons. Instead, an isoform of ISPa21 (96% homology) was inserted into the attC site of the smr cassette between the core sites 1L and 2L (Figure 1b). Similar insertions of ISPa21 have been described in the attC sites of the blaGES and aadB cassettes in In10915 and the aadA7 cassette in In163 (accession no. AY660529 [GenBank] ). These findings support the notion that the attC-conserved motif CAATTCA( )TCC( ) is a specific target for ISPa21.15
Association of the In416-containing region with a novel transposon
A transposon sequence, designated Tn8802, was inserted into the IRL of ISPa21. Tn8802 carried an arsenic resistance operon (99% homologous to the ars operon from plasmid R46) (accession no. AY046276 [GenBank] ) (Figure 1c) that was probably functional as suggested by the decreased susceptibility of E. coli (B1) (Figure 1a) to sodium arsenite (data not shown). Tn8802 included a tnpR resolvase gene identical to that found downstream of the ars operon in R46 (namely, TnARS).16 The adjacent transposase, however, was identical to tnpA of Tn2* located on plasmid pRMH760.13 The 163 bp junction between tnpR and tnpA appeared as a hybrid of the res sequences of the two aforementioned elements (Figure 1b and c). Tn8802 was flanked by two 38 bp IRs exhibiting seven mismatches. The IRL of Tn8802 was identical to that flanking the ars operon of R46, whereas the IRR was identical to that of Tn2*. Taken together with the lack of direct repeats in the flanks of Tn8802, the sequence characteristics of this element suggested that it was a Tn2–TnARS hybrid probably generated by recombination at the resI site of these transposons.
The IRR of Tn8802 was merged with another class 1 integron containing a four-cassette array (aadB, orfE-like, orf416 and cmlA7) similar to In-t4,4 conferring resistance to additional non-ß-lactam antibiotics, including chloramphenicol, as observed in E. coli CON036A (data not shown). Insertion of Tn8802 caused a deletion of the 3'-CS including 502 bp of the 3' end of sul1 that was inactive, as also shown by the susceptibility of the pACYC184-5G3-carrying clones (Figure 1a) to sulphonamides. Nevertheless, pCC416 mediated resistance to sulphonamides. In fact, PCR analysis revealed the presence of a sul2 determinant on pCC416, indicating existence of an additional resistance-encoding region that was not identified in this study. In-t4 was previously described in the blaCMY-2-carrying plasmid from Salmonella enterica serotype Typhimurium, strain 2039, isolated in the USA in 19994 and also showing the Tn2 at the 3'-CS (Figure 1c, EMBL accession no. AJ487033 [GenBank] ). The finding that these plasmids share the same integron and belong to the same Inc group (IncA/C2)3 strongly suggests that they belong to a common lineage.
The blaCMY-4-containing region of pCC416
The similarity of pCC416 with the CMY-2-positive IncA/C2 plasmids from the USA prompted us to determine the sequence surrounding blaCMY-4 on the pCC416 plasmid. The genetic context of blaCMY-4 (Figure 2b) was characterized by sequencing of the 11 251 bp insert of clone C12. The blaCMY-4 gene occurred as part of a Citrobacter freundii-derived sequence of 2825 bp that also comprised blc, sugE and 
ecnR and was preceded by an ISEcp1. Upstream of ISEcp1, an ORF encoding a putative protein similar to TraA (89% identity) that is involved in conjugative pilus assembly, was identified.17,18 A 6485 bp sequence downstream of ecnR included ORFs of unknown function, a disulphide bond isomerase gene (dsbC) and 41 bp from the 5' end of traC. The first part of the structure, 4883 bp in size, was similar to the respective region of the CMY-2-encoding plasmids (types A and C) from Salmonella in the USA, reported by Giles et al.19 The entire CMY-4-encoding segment (11 047 bp) of pCC416 differed by only 21 nucleotides from the respective sequence of the type I element of the CMY-encoding plasmids from Salmonella and E. coli isolates (from nt 1 to nt 11 046 in accession no. DQ164214
[GenBank]
), but lacked the right-hand ISEcp1, thus resembling the type II structure.20
It was not possible to localize the position of the blaCMY locus with respect to the In416 region. Long-range PCR assays using combinations of primers designed on ß-lactamase, transposase and integrase genes (Table 1) were negative, suggesting that the VIM-4- and CMY-4-encoding sequences most likely resided in distant loci. This notion is compatible with the large size of the pCC416 plasmid, estimated to be 
120 kb (data not shown).
pCC416 represents an efficient vector for the en bloc transfer of resistance to most clinically useful antibiotics. In particular, pCC416 carried the MBL gene blaVIM-4 and the AmpC-like ß-lactamase gene blaCMY-4 that were located in two distinct regions. The sequencing data suggest that pCC416 derived from the IncA/C2 CMY plasmid through acquisition of a segment containing a novel blaVIM-4 integron. In416 was closely related to class 1 integrons carrying blaVIM-1 alleles from various Gram-negative species isolated mainly in Europe suggesting a common origin. In416 was associated with a Tn1696 module as the prototype In4 integron, whereas in both In-e5417,8 and pPBH01,9 the Tn1696 was not found, possibly due to IS26 insertions. Nevertheless, In-e541 also belonged to the In4 type, further supporting the genetic relatedness of these structures. The presence of a partial Tn1696 sequence indicated that In416 might be more closely related to the hypothetical parental structure. The occurrence of similar VIM-encoding integrons in diverse genetic locations indicates mobilization mechanisms. In the case of In-e541 and pPBH01, mobilization might have been mediated by IS26. However, in pCC416, the characteristics of the sequence intervening In416 and In-t4 suggest a process involving at least one recombination event at the res sites of a Tn2 of a CMY plasmid. These findings underscore the variety of mechanisms implicated in the evolution of multiresistant plasmids.
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None to declare.
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Present address. Laboratoire de Radioécologie et d'Ecotoxicologie, Institut de Radioprotection et de Sûreté nucléaire, DEI/SECRE/LRE, Bât 186, BP 3, F-13115 Saint Paul Lez Durance Cedex, France. 
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Thanks are due to Gianluca Lentini for technical support. This work was supported by grants from the European Commission (MEBEL project, contract no. HPRN-CT-2002-00264; COBRA project, contract no. LSHM-CT-2003-503335; and DRESP2 project LSHM-CT-2005-018705).
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